Ivancich A, Lutz M, Mattioli T A
Departement de Biologie Cellulaire et Moleculaire, CEA and CNRS URA 2096, CEA/Saclay, Gif-sur-Yvette, France.
Biochemistry. 1997 Mar 18;36(11):3242-53. doi: 10.1021/bi962483i.
We have reexamined the temperature dependence of resonance Raman (RR) spectra of the bacteriochlorin cofactors bound to reaction centers from Rhodobacter sphaeroides. Three types of resonant excitations were performed, namely, Soret band, bacteriopheophytin Qx-band, and near-infrared, Qy-band (pre)resonances. Sample temperature was varied from 300 to 10 K. In both Soret-resonant and Qy-preresonant Raman spectra, the ca. 1610-cm(-1) band corresponding to a bacteriochlorophyll CaCm methine bridge stretching mode is observed to increase in frequency by 4-6 cm(-1) as temperature is decreased from 300 to 15 K. This upshift is interpreted as arising from a change in conformation of the bacteriochlorophyll macrocycles. It may be nonspecific to the protein-bound cofactors, since a similar 4-cm(-1) upshift was observed in the same temperature range for BChl a in solution. Qx-resonant Raman spectra of either of the two bacteriopheophytin (BPhe) cofactors were obtained selectively using excitations at 537 and 546 nm. No significant frequency shift was observed for the CaCm stretching mode of BPheL between 200 and 15 K. We conclude, at variance with a previous report, that the macrocycle of the BPheL primary electron acceptor does not undergo any significant conformational change in the 200-15 K temperature range. Qy-preresonant excitation of RCs at 1064 nm provided selective Raman information on the primary electron donor (P primary). The stretching frequencies of the two conjugated keto and acetyl carbonyl groups of the M-branch primary donor BChl cofactor (P(M)) did not significantly change between 300 and 10 K. In contrast the keto carbonyl stretching frequency of cofactor P(L) was observed to upshift by 5 cm(-1), while its acetyl carbonyl frequency downshifted by 2 cm(-1). The latter shift indicated that the strong H-bond between the acetyl group of P(L) and His L168 may have slightly strengthened at 10 K. Excitation at 1064 nm of chemically oxidized RCs selectively provided RR spectra of the primary donor in its radical P.+ state. These spectra can be interpreted as a decrease of the localization of the positive charge on P(L) from 78% to 63% when the temperature decreased from 300 to 10 K resulting in a more electronically symmetric dimer. Possible origins of the temperature dependence of the positive charge delocalization in P.+ are discussed.
我们重新研究了与球形红细菌反应中心结合的细菌叶绿素辅因子的共振拉曼（RR）光谱的温度依赖性。进行了三种类型的共振激发，即Soret带、细菌脱镁叶绿素Qx带和近红外Qy带（预）共振。样品温度在300至10K之间变化。在Soret共振和Qy预共振拉曼光谱中，当温度从300K降至15K时，对应于细菌叶绿素CaCm次甲基桥伸缩模式的约1610-cm⁻¹带的频率增加了4-6 cm⁻¹。这种频率上移被解释为细菌叶绿素大环构象的变化。这可能对与蛋白质结合的辅因子不具有特异性，因为在相同温度范围内，溶液中的BChl a也观察到了类似的4-cm⁻¹频率上移。使用537和546nm的激发光选择性地获得了两种细菌脱镁叶绿素（BPhe）辅因子中任一种的Qx共振拉曼光谱。在200至15K之间，未观察到BPheL的CaCm伸缩模式有明显的频率 shift。我们得出结论，与先前的报告不同，在200至15K的温度范围内，BPheL初级电子受体的大环没有发生任何明显的构象变化。在1064nm处对反应中心进行Qy预共振激发，提供了关于初级电子供体（P原初）的选择性拉曼信息。M分支初级供体BChl辅因子（P(M)）的两个共轭酮基和乙酰羰基的伸缩频率在300至10K之间没有明显变化。相比之下，观察到辅因子P(L)的酮羰基伸缩频率上移了5 cm⁻¹，而其乙酰羰基频率下移了2 cm⁻¹。后一种 shift 表明，P(L)的乙酰基与His L168之间的强氢键在10K时可能略有增强。在1064nm处对化学氧化的反应中心进行激发，选择性地提供了处于自由基P⁺状态的初级供体的RR光谱。这些光谱可以解释为，当温度从300K降至10K时，P(L)上正电荷的局域化从78%降至63%，导致二聚体在电子上更加对称。讨论了P⁺中正电荷离域的温度依赖性的可能起源。
